A rapid and visual loop-mediated isothermal amplification assay to detect Leifsonia xyli subsp. xyli targeting a transposase gene.

UNLABELLED: Leifsonia xyli subsp. xyli (Lxx), causal organism of ratoon stunt (RSD), does not produce any reliable internal or external symptoms on sugarcane. Its detection on a large scale is solely based on microscopic and serological methods. These methods require well-equipped laboratories, are time consuming and are not feasible for near-field detection of Lxx. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of Lxx without the use of sophisticated equipment. To the best of our knowledge, this is the first report on the detection of Lxx in 30 min via an isothermal amplification method at 65°C. A transposase gene, ISLxx5, was used to design a set of six primers specifically targeting eight genomic sequences. The xylem sap was used as template, thus circumventing the need to isolate pure genomic DNA. The positive reactions were visually detected through a colour change of hydroxynaphthol blue (HNB) from violet to light blue, thus, eliminating the need for gel electrophoresis. The LAMP method was 10 times more sensitive than serological detection and as sensitive as immunofluorescence microscopy (IFM). The simplicity and sensitivity of the ISLxx5 LAMP assay makes it suitable for near-field diagnosis of RSD. SIGNIFICANCE AND IMPACT OF THE STUDY: Detection of Leifsonia xyli subsp. xyli (Lxx) on a large scale is based on serological assays such as evaporative-binding enzyme-linked immunoassay (EB-EIA). These methods are time consuming and require well-equipped laboratories. This study presents the development of a loop-mediated isothermal amplification (LAMP) assay which allows detection of Lxx in 30 min at 65°C, using xylem sap as the template. The assay requires minimal laboratory equipment and could be used at near farm conditions, thus saving time and money required to transfer samples from remote areas to diagnostic laboratories. The LAMP method shows potential as an alternative detection method for RSD.

Beauveria brongniartii on white grubs attacking sugarcane in South Africa.

Beauveria brongniartii (Saccardo) Petch fungal infections were observed on the melolonthid Hypopholis sommeri Burmeister (Coleoptera: Scarabaeidae) at two sites (Harden Heights and Canema) in the sugarcane producing area of the northern KwaZulu-Natal Midlands of South Africa. To initially identify the disease-causing fungus, 17 different fluorescently-labelled microsatellite PCR primers were used to target 78 isolates of Beauveria spp. DNA. Microsatellite data resolved two distinct clusters of Beauveria isolates which represented the Beauveria bassiana s.s. (Balsamo) Vuillemin (17 isolates) and B. brongniartii (60 isolates) species groups. These groupings were supported by two gene regions, the nuclear ribosomal Internal Transcribed Spacer (ITS) and the nuclear Bloc gene of which 23 exemplar Beauveria isolates were represented and sequenced. When microsatellite data were analysed, 26 haplotypes among 58 isolates of B. brongniartii were distinguished. Relatively low levels of genetic diversity were detected in B. brongniartii and isolates were shown to be closely related. No genetic differentiation was observed between the Harden Heights and Canema populations; they thus may be considered one, structured and fragmented population over a distance of 5.5 km. Historically high levels of gene flow from swarming H. sommeri beetles is the proposed mechanism for this observed lack of genetic differentiation between populations. Microsatellite analyses also showed that B. brongniartii conidia were being cycled from arboreal forest to subterranean sugarcane habitats and vice versa in the environment by H. sommeri life stages. This is the first record of this species of fungus infecting H. sommeri larvae and adults in South Africa.

Towards a rapid near-infrared technique for prediction of resistance to sugarcane borerEldana saccharina walker (Lepidoptera: Pyralidae) using stalk surface wax.

Multiple regression predictive models based on data acquired by near-infrared (NIR) spectrophotometry suggest that stalk surface wax components contribute towards resistance toEldana saccharina Walker in sugarcane. At least 35 sugarcane clones of known resistance were required to calibrate a predictive model that accounted for approximately 54% of the variation in resistance toEldana. Wavelengths chosen in multiple regression models suggest that alcohols and carbonyls are important in the wax contribution. Through the use of wax fractionation and gas chromatography, a high alcohol/aldehyde ratio and shorter carbon chain length appears to be associated with resistance. The use of NIR in the screening of wild germplasm and the early screening of breeding material for resistance, without prior knowledge of the biochemical mechanisms involved, is an exciting prospect. However, cause-and-effect relationships remain to be shown.